QC Report

	general
Report generated at	2025-02-25 00:22:59
Title	Pancreas
Description	ATAC-seq data from Human ENCODE pancreas data processed in 2025
Pipeline version	v2.2.0
Pipeline type	atac
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	138334834	425693838
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	135923011	413925919
Mapped Reads (QC-failed)	0	0
% Mapped Reads	98.3	97.2
Paired Reads	138334834	425693838
Paired Reads (QC-failed)	0	0
Read1	69167417	212846919
Read1 (QC-failed)	0	0
Read2	69167417	212846919
Read2 (QC-failed)	0	0
Properly Paired Reads	133009924	406375972
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	96.2	95.5
With itself	134206540	409714240
With itself (QC-failed)	0	0
Singletons	1716471	4211679
Singletons (QC-failed)	0	0
% Singleton	1.2	1.0
Diff. Chroms	128713	233817
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	59783571	180149877
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	9261193	25493700
Paired Optical Duplicate Reads	674269	2702838
% Duplicate Reads	15.4912	14.1514

Filtered with samtools flag 1804 (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2
Rn = Number of Non-mitochondrial Reads	124125942	397055770
Rm = Number of Mitochondrial Reads	12362057	17962177
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.09057248322616261	0.0432804825185066

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	98571138	307055396
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	98571138	307055396
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	98571138	307055396
Paired Reads (QC-failed)	0	0
Read1	49285569	153527698
Read1 (QC-failed)	0	0
Read2	49285569	153527698
Read2 (QC-failed)	0	0
Properly Paired Reads	98571138	307055396
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	98571138	307055396
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	0	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2
Fraction of reads in NFR	0.3145150198755472	0.35047792539723627
Fraction of reads in NFR (QC pass)	False	False
Fraction of reads in NFR (QC reason)	out of range [0.4, inf]	out of range [0.4, inf]
NFR / mono-nuc reads	1.209615409425759	1.5135908362866812
NFR / mono-nuc reads (QC pass)	False	False
NFR / mono-nuc reads (QC reason)	out of range [2.5, inf]	out of range [2.5, inf]
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	True	True
Presence of Di-Nuc peak	True	True

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.5973919668047253	0.4024372657499235
Fraction of Reads in blacklist regions	0.0007360572422325083	0.0012917115451050402
Fraction of Reads in promoter regions	0.3444767473416001	0.1737267825119087
Fraction of Reads in enhancer regions	0.31561432312975835	0.32585977743247346

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	55558177	174037843
Distinct Fragments	49493558	154341567
Positions with Two Read	4624398	14686568
NRF = Distinct/Total	0.890842	0.886828
PBC1 = OneRead/Distinct	0.893438	0.890795
PBC2 = OneRead/TwoRead	9.562198	9.36139

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	217013	139090
N1	205373	148487
N2	257215	169303
Np	266980	184782
N optimal	266980	184782
N conservative	217013	139090
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.2302488790994088	1.3285067222661586
Self Consistency Ratio	1.2524285081291113	1.1401873564689164
Reproducibility Test	pass	pass

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2
Number of peaks	242579	299607

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0
25 percentile	311.0	292.0	629.0	412.0
50 percentile (median)	641.0	560.0	1005.0	751.0
75 percentile	1095.0	1044.0	1402.0	1232.0
Max size	4361.0	3711.0	3680.0	3680.0
Mean	753.274665160628	720.6006868998388	1050.0259711443755	865.589961794891

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	27.23982136437038	15.94762653291641

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.12316706891407908	0.24041904351035
Synthetic AUC	0.49630581030942345	0.49790422743066864
X-intercept	0.12016040845782781	0.09267466709847408
Synthetic X-intercept	0.0	0.0
Elbow Point	0.8767049895746497	0.7768695161957767
Synthetic Elbow Point	0.5002546515010872	0.5021212278421999
Synthetic JS Distance	0.5876073884619921	0.38480852769397506

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.534280014094998	0.3161604461756471	0.5232764681427038	0.3090736956141946	0.5210860307017259	0.30889302463194623	0.3748068783882861	0.36701406044105556	0.3652654210499228

FRiP for overlap peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.34435048571058224	0.519842116462123	0.3037588631075547	0.36550431634238206

FRiP for IDR peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.2993005852028408	0.48538830910118946	0.2653192455214172	0.3311399939137118

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates